Antithrombin (AT), a plasma serine protease inhibitor (serpin), inactivates factor Xa and thrombin by a branched pathway, suicide substrate inhibition mechanism in which an initial reversible Michaelis complex is converted into a tetrahedral or an acyl-enzyme intermediate similar to the normal reaction of these enzymes with their true substrates. In contrast to true substrates, however, a conformational change in the reactive site loop of the serpin leads to insertion of the loop into the A-beta-sheet of the molecule. This results in distortion of the catalytic machinery so that the intermediate is deacylated very slowly and, therefore, is trapped as a kinetically stable, irreversible complex. Unlike AT, binding of the non-serpin inhibitor, tissue factor pathway inhibitor, to factor Xa traps the protease as a reversible, high affinity Michaelis complex. AT is relatively inactive unless it binds to the heparin-like glycosaminoglycans, which are either present in the microvasculature or therapeutically administered for prophylaxis and treatment of venous thrombosis. The low inhibitory activity of AT is due to partial preinsertion of the reactive site loop of AT into the A-beta-sheet of the molecule. Heparin binding to AT induces a conformational change in the reactive site loop of the serpin that causes the expulsion of this inserted region thereby conferring a canonical conformation for the loop that is complimentary to the active site pocket of protease. We have prepared several serpin and protease mutants to investigate the following five Aims: 1) determine the contribution of the catalytic Ser195 of the proteases in binding to the serpin and non-serpin inhibitors; 2) determine whether the AT-protease complexes are trapped as tetrahedral or acyl-enzyme intermediates; 3) determine how heparin might catalyze the AT inactivation of factor Xa in the prothrombinase complex; 4) determine whether there is a heparin binding "exosite" on factor Xa, analogous to that in thrombin; and 5) determine the structural basis for the partial loon preinsertion and the resulting non-canonical conformation of the reactive site loop of the serpin.